Tuberculosis is one of the leading causes of death worldwide from an infectious disease. Isoniazid is a front line antibiotic used in the treatment of tuberculosis. The Mycobacterium tuberculosis heme catalase/peroxidase KatG is responsible for activating isoniazid to a reactive biocidal species. Antibiotic resistance to isoniazid is a growing concern and can occur by deletions or point mutations in the katG gene. One of these, a serine-to-threonine substitution at position 315, KatG(S315T) is found in about 50 percent of clinical isolates resistant to isoniazid. Our research seeks to understand the mechanism of isoniazid activation and molecular basis for drug resistance caused by the S315T and other point mutations in KatG using biochemical, genetic, and spectroscopic techniques. KatG will be purified from recombinant and native sources and the activities and spectroscopic properties compared. Besides a catalase/peroxidase activity, KatG can also catalyze several other reactions including Mn2+ peroxidase, P450-like oxygenase, and peroxynitritase activities. Which of these or possibly other redox reactions are responsible for isoniazid oxidation and activation will be tested using wild-type and mutant KatG proteins to investigate the mechanism of activation. Genetic methods investigating the role of superoxide in isoniazid activation are proposed. Optical, EPR, NMR, and resonance Raman spectroscopies are revealing subtle differences in the heme active site of wild-type KatG and KatG(S315T). These spectroscopic techniques will be applied to various forms of wild-type and mutant enzymes in order to elucidate the molecular basis for reactivity toward isoniazid and the reduced rate of isoniazid oxidation by KatG(S315T). NMR relaxation measurements and x-ray crystallography will be used to map the isoniazid binding site on both enzymes to determine whether subtle differences in distance and/or orientation are responsible for the reduced turnover of drug by the mutant enzyme. Steady-state and rapid kinetic techniques will follow ligand-binding rates to the heme iron and the formation and decay of reactive intermediates in the catalytic cycle for both wild-type KatG and KatG(S3 1ST) to determine whether the S3 1ST mutation affects one of the steps in the catalytic cycle. EPR spectroscopy and spin-traps are being used to trap reactive intermediates. The stable products formed in this reaction are being characterized by mass spectrometry to reveal chemical information about the nature of intermediates in the reaction of isoniazid oxidation by KatG. Site directed mutagenesis of residues also implicated in isoniazid resistance other than S3 15 will be generated and the effects on enzyme activities and spectroscopic properties examined to determine structural information about the mutant enzymes. Analogs of isoniazid will be used as additional biochemical and spectroscopic probes of the reaction mechanism and molecular basis for drug resistance.